PROJECT SUMMARY Despite substantial gains in survival of preterm infants, concerns remain regarding the significant neurological morbidity and long-term adverse outcomes related to insults on the immature immune and brain-gut-microbiota systems affected by painful/stressful early life experience during the neonatal intensive care (NICU) stay. Our preliminary K23 results show that cumulative pain/stress events are significantly associated with higher abundance of gut Enterobacteria (Phylum: Proteobacteria), a characteristic pattern of dysbiosis, which may contribute to neurodevelopmental deficits during the NICU stay. In light of these results, the primary hypothesis driving this research is that cumulative pain/stress experienced in early life combined with gut dysbiosis and specific genetic susceptibilities increase the risk of neurodevelopmental morbidity in preterm infants during infancy and early childhood. A prospective longitudinal design will be used to examine: 1) the impact of cumulative pain/stress events in the NICU along with gut microbiome development on infant neurodevelopmental outcomes over the short- (NICU stay) and long-term (follow-up); 2) interaction effects of host genetics, gut microbiome, and early life pain/stress events on infant neurodevelopmental outcomes, while controlling for sex, feeding and other environmental factors over time; and 3) the impact of different levels of pain/stress experiences on the gut microbiome and neurodevelopment outcomes as well as other growth parameters using twin- pairs. The proposed 4-year project will recruit and follow 200 preterm infants (160 infants in the final analysis considering the attrition) during NICU hospitalization and until 18-24 months corrected age (CA). Primary measures in the NICU include daily pain/stress events (NICU Infant Stressor Scale), gut microbiome patterns and function (stool sample: twice/week; 16S rRNA gene and metagenomic sequencing), host genetics (whole exome sequencing to identify genetic variants that effect neuro-gut-immune signaling), autonomic responses (weekly; heart rate variability) and neurodevelopmental outcomes (at 36 weeks CA; NICU Network Neurobehavioral Scale). At follow-up visits, gut microbiome, neurodevelopmental outcomes, including pain sensitivity will be measured at 4, 8-12, and 18-24 months CA.